There is a continuing need in medical practice, reserch and diagnostic procedures for rapid and accurate detection or quantification of biological and chemical substances which are present in biological fluids, cells or tissues. For example, the presence of drugs, hormones, steroids, polypeptides, nucleotides, prostaglandins, proteins, carbohydrates or infectious organisms (bacteria, fungi or viruses) in biological specimens has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
For example, organisms classified as gram positive bacteria, such as group specific Streptococcus, are known to be pathogenic in humans. For instance, Group A organisms are primarily responsible for causing B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac sequelae, glomerulonephritis, septic sore throat and puerpueral sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence at an early stage so that an appropriate course of treatment can be pursued. In most cases, the diagnostic tests require several hours, or at least up to 30 minutes. Even this limited wait may be intolerable in many instances where the practitioner has many waiting patients, and the patients themselves can not wait for the diagnosis without considerable cost, inconvenience or discomfort.
To provide diagnostic determinations, various methods have been devised for isolating and identifying biological or chemical substances employing specific binding reactions between the substance to be detected (identified as a "target ligand" or simply "ligand" herein) and receptors (compound which specifically react or bind with that substance). This reaction between a ligand and its corresponding receptor is known as a specific binding reaction. Where either the ligand or receptor is an antibody, the reaction is known as an immunological reaction. More than one ligand or receptor may participate in each reaction.
Such reactions are detected in a number of ways. Generally, one or more participants of the specific binding reaction is detectably labeled. That is, it is either chosen because it is inherently detectable, or a detectable moiety (for example, an enzyme, radioisotope, chromogen or fluorogen) is incorporated therein in some manner.
Furthermore, it is often necessary in detecting the products of the specific binding reactions described above that the products be insolubilized in some manner and separated from unreacted materials. Various insolubilizing means have been used including particles, the sides of containers such as test tubes, thin films and others known in the art. In some assays and diagnostic devices, a receptor for the ligand is bound to a porous membrane or filter, such as described in related U.S. Pat. Nos. 4,632,901 (issued Dec. 30, 1986 to Valkirs et al) and 4,727,019 (issued Feb. 23, 1988 to Valkirs et al). These membranes or filters serve both to insolubilize the resulting complex as well as to separate it from uncomplexed materials by filtration.
PCT Publication 87/03690 (published June 18, 1987) also describes the use of a porous filtration membrane in an immunoassay wherein an insolubilized immunocomplex is trapped in the membrane as fluid drains through. The insolubilized immunoreagents can be incorporated into the membrane prior to the assay for storage. A similar solid phase membrane system is described in E. P. Publication 200,381 (published Nov. 5, 1986). Microspheres having receptor molecules attached thereto are entrapped within a porous matrix. A similar diagnostic test for Streptococcus A antigen has been commercially available for about two years.
However, assays carried out using entrapped reagents lack sufficient sensitivity and detectability of the complex in order to provide highly successful assays in the marketplace.
A diagnostic test kit for Streptococcus A determination, which has been marketed by Hybritech, Inc. for about two years as the Tandem.RTM. Icon.RTM. Strep A test, has particulate immunoreagents coated on top of a membrane. However, this test kit has disadvantages, namely low sensitivity for samples with low colony counts and slow assay dye kinetics.
Moreover, it has been found that specific binding reagent affixed to the microporous substrate lacks sufficient keeping stability for the lengthy times often required for manufacture, shipping and storage prior to use. If the reagent is used relatively soon after manufacture, stability is not a serious problem. However, a diagnostic test device shipped a considerable distance from the site of manufacture (for example, remote or underdeveloped areas of the world) must have acceptable refrigerator and room temperature keeping stability over several weeks or months to assure accurate and sensitive results when it is eventually used. Such a product useful for various diagnostic assays, and particularly to detect Streptococcal antigens, would be greatly welcome in the industry.